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DOI: 10.1002/qaj.365
GLP-compliant Assay ValidationStudies: Considerations forImplementation of Regulations andAudit of Studies
Henry Li*, Garreth Sharpy, Connie Pilkington, Dominique Pifat and Steve Petteway
Talecris Biotherapeutics, 85 TW Alexander Drive, Research Triangle Park, NC 27709, USA
Summary
Bioanalytical methods used in nonclinical safety studies are validated to demonstratethat the methods are reliable and reproducible for the intended use. Analyticalmethod validation studies should be designed according to regulatory guidance onmethod validation. A cell-based assay is described to discuss fundamental validationparameters, such as repeatability, immediate precision, accuracy, and linearity. AGood Laboratory Practice (GLP) compliance program provides a useful platform forensuring data integrity in assay validation studies. A good understanding of themethod validation concepts and regulatory requirements will help Quality AssuranceUnit (QAU) auditors focus on key areas and be more effective when auditing assayvalidation protocols, final reports, and associated data. Copyright # 2006 John Wiley& Sons, Ltd.
Key Words: assay validation; audit/inspection; Good Laboratory Practice (GLP); QualityAssurance
Introduction
It is scientifically sound and often required to
validate the bioanalytical methods used in
support of Good Laboratory Practice (GLP)-
compliant pharmacology/toxicology studies,
other nonclinical safety studies such as pathogen
safety studies and testing of human samples to
support clinical trials. To ensure quality, such
validation studies are often conducted in com-
pliance with GLP principles, even though this is
not required by the regulations. GLP concepts,
such as the requirements for protocol, final
report, and conduct of a nonclinical laboratory
study can be directly implemented in an assay
validation study. However, there are challenges
that require careful interpretation of the regula-
tions.
The Quality Assurance Unit (QAU) is respon-
sible for monitoring the assay validation studies
for GLP compliance and conducting protocol,
‘in-life’, and final report audits. To effectively
approach these audits, it is helpful for auditors
to be familiar with assay procedures and
validation requirements. Data generated from
an assay validation study can be complex and
several critical compliance elements are unique
to assay validation studies. It is important not to
lose sight of key areas when conducting quality
audits.
*Correspondence to: Henry Li, Talecris Biotherapeutics,85 TW Alexander Drive, Research Triangle Park, NorthCarolina 27709, USA. E-mail: [email protected] address: Ethicon Inc., Somerville, NJ, USA.
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In this paper, a validation study of a cell-based
biological assay (porcine parvovirus assay (PPV))
is used as an example to discuss experience in
implementation of GLP compliance in assay
validation studies. Basic assay validation con-
cepts and regulatory requirements are discussed.
Points for consideration for auditing assay
validation studies, including those conducted in
compliance with GLP, are presented.
Regulatory Expectations on AssayValidation
Although assay validation is not directly
addressed in GLP regulations, it appears that
the Food and Drug Administration (FDA)
encourages the use of validated methods. In a
recent Warning Letter issued on 21 December
2004 to Nelson Laboratories Inc. [1] by the
Center for Devices and Radiological Health
(CDRH), FDA cited that [t]he QA unit did not
adequately monitor each study, as required by
21 Code of Federal Regulations (CFR)
Part 58.35(a). For example, ‘‘[t]he current test
methods, which are described in SOP[. . .], have
not been fully validated’’. It is not clear whether
this finding signals a significant shift in the
regulatory interpretation of the GLP regulations;
however, this finding does suggest that validat-
ing analytical procedures used in GLP studies is
the right direction moving forward. It is also
worth pointing out that in this case the agency
put the burden on the QAU to ensure that test
methods are validated.
The FDA guideline ‘Guidance for Industry:
Bioanalytical Method Validation’ provides cur-
rent regulatory expectations for the compliance
level of bioanalytical testing laboratories [2]. It
states that the analytical laboratory conducting
pharmacology/toxicology and other preclinical
studies for regulatory submissions should adhere
to FDA’s GLP regulations (21 CFR Part 58) and
sound principles of quality assurance through-
out the testing process.
The development of biopharmaceuticals
requires accurate and reliable analytical proce-
dures. The extent to which assay validation is
required has been extensively discussed in the
industry. It is often expected that the analytical
procedures used under the auspices of GLP or
current Good Manufacturing Practice (cGMP)
are validated [3]. Recently, at the third CMC
Strategy Forum held on the campus of the
National Institute of Standards and Technolo-
gies (NIST) [4], which was attended by both the
industry and government representatives, it was
concluded that complete assay validation is
required for GLP study assays, lot release assays,
raw material testing, in process testing, excipient
testing, and stability methods for defining
expiration dates/hold times. It was emphasized
that some assays, especially those used for GLP
animal toxicology or related safety studies, are
expected to be validated, despite such studies
occurring very early on in the drug development
process [4].
Basic Assay Validation Concepts
Types of method validation studies
There are several regulatory guidelines on assay
validation [2, 5–7]. Depending on the intended
use of a particular bioanalytical method and the
stage of the method development, types and
levels of the validation required can be quite
different. The types of method validation studies
can be classified into three general categories.
* Full validation is often required when new
bioanalytical methods are developed; the
method is used for detecting a new drug
entity; or metabolites are added to an existing
method for quantification [2].* Partial validation is necessary when already
validated bioanalytical methods are modified.
The validation exercise, depending on the
intended use of the method, ‘‘can range from
as little as one intra-assay accuracy and
precision determination to a nearly full
validation’’ [2].* ‘‘Cross-validation is a comparison of valida-
tion parameters when two or more bioanaly-
tical methods are used to generate data within
the same study or across different studies’’ [2].
GLP-compliant Assay Validation Studies 93
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Viral infectivity assay
Biological assays based on cell culture and
animals are performed in complex biological
systems. These assays are often cumbersome and
highly variable. The PPV infectivity assay is used
here as an example to discuss assay validation
parameters and requirements. This assay is used
in GLP-compliant viral clearance studies [8,9] to
validate the capability of biologics manufactur-
ing processes to remove or inactivate PPV. The
results obtained from these safety studies will be
used for Investigational New Drug application
(IND), Biological License Application (BLA)
and to support changes to commercial produc-
tion processes [10]. This typical, cell-based viral
infectivity assay is schematically illustrated in
Figure 1.
Fundamental assay parameters
The objective of assay validation is to demon-
strate the assay is suitable for the intended use.
The requirements for the performance of the
PPV infectivity assay used in viral clearance
studies are included in the regulatory guidelines
[8,9]. The approach taken to the validation of
PPV infectivity assay is based on the recommen-
dations contained in the regulatory guidelines on
assay validation including assay validation
design and other considerations [2, 5–7]. How-
ever, this biological assay possesses unique
characteristics. For example, the readout of the
assay, cytopathic effect as a result of lysis of
infected cells, requires interactions of virus with
live cells, including viral entry into cells and
subsequent replication of virus inside the cells,
leading to cell death. Some assay acceptance
criteria were pre-determined based on the
complexity of the assay and might not be
consistent with those stated for analytical
methods in the FDA guideline [2]. Several
critical assay validation parameters validated
are briefly presented below.
RepeatabilityThe closeness of the results between a series
of measurements of a single sample obtained
by a single analyst under the same operating
conditions over a short interval of time. Repeat-
ability is also called as intra-assay precision.
Repeatability is often mathematically expressed
Day 1• Make serial half-log dilutions of each virus containing sample• Add each dilution to 8 replicate wells (one row) of the indicator cell plate with an appropriate cell density (40-60% confluence)• Let virus adsorb to cells for about 1 hour at 37˚C• Remove inocula from plates; replace with appropriate medium• Incubate plates for specified time at 37° C
Day 7-10• Examine plates microscopically for cytopathic effect
Titer Calculation
Cell Control
SampleDilution
Adsorption to cell
8/88/88/88/88/85/84/82/81/8
0/80/8
0/8
Incubation
Dilution of sample
High
Low
NegativePositive
Figure 1. Schematic diagram of PPV infectivity assay
94 H Li et al.
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as percent of Coefficient of Variation (% CV)
of a series of measurements and is figuratively
illustrated in Figure 2. The repeatability of
the PPV infectivity assay was evaluated at three
PPV concentrations, high (undiluted), mid
(1:100 dilution of the high concentration), and
low (1:100 dilution of the mid concentration)
levels of PPV. A single analyst titrated three
samples in triplicate and performed the repeat-
ability experiment totally three times on
three separate days (Week 1, 2, and Week 3)
to yield nine determinations for each concen-
tration.
Intermediate precisionIntermediate precision is assay-to-assay varia-
bility within laboratories such as different
analysts, different instruments and different
days. The intermediate precision is also illu-
strated in Figure 2. During the first two
repeatability experiments, an additional analyst
also independently performed the PPV assay on
three PPV concentrations (high, mid, and low).
The results generated by two analysts were
analysed and compared.
AccuracyAccuracy is the measure of the closeness or
agreement between the measured value and the
true value or an accepted reference value of an
analyte (e.g. PPV in this study). Accuracy is
often expressed as percent of true or actual value
(Figure 2). In this assay validation study, the
PPV stock was serially diluted (1:10) to generate
nine PPV concentrations. The concentrations of
diluted PPV samples were determined based on
the calculation using the original titer of the PPV
stock and represented the ‘true values’ of PPV.
Each sample was assayed and the experiment
was repeated on two separate days in two
weeks. The measured values and the true values
were used to calculate the accuracy of the PPV
assay. The results from these experiments were
also used to determine the linearity, quantifica-
tion limit, and detection limit.
LinearityThe linearity of a method is its ability within a
given range to elicit results that are directly, or
by a well-defined mathematical transformation,
proportional to concentration of an analyte (e.g.
PPV in this validation study) in the sample.
Linearity is generally reported as the variance of
the slope of the regression line. In Figure 2,
the linear regression coefficient R2 is used.
The experimental design is described in the
section for accuracy.
Not Precise Precise & Accurate
Intermediate Precision
Not Precise
Repeatability
Precise Accurate Not Accurate
Accuracy
0
1
2
3
4
5
6
7
8
Log
PP
V T
iter
of O
bser
ved
0 1 2 3 4 5 6 7 8
Log PPV Titer of Expected
Linearity
Analyst 2Analyst 1
Figure 2. Repeatability, intermediate precision, accuracy, and linearity
GLP-compliant Assay Validation Studies 95
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Quantification limitThis refers to the lowest level of an analyte (e.g.
PPV in this validation study) in a sample that
can be quantitatively determined with suitable
precision and accuracy. The experimental design
for determining quantification limit is described
in the section for accuracy.
Detection limitThe limit of detection is the lowest concentra-
tion of analyte (e.g. PPV in this validation study)
that can be reliably detected by the method and
is expressed in terms of the concentration of
analyte in a sample. The experimental design is
described in the section for accuracy.
SelectivitySelectivity is the ability of the assay to differ-
entiate and quantify the analyte (e.g. PPV) in the
presence of other components in the sample. If
the method is intended to quantify more than
one analyte, each analyte should be tested to
ensure that there is no interference. In this study,
selectivity is defined as the ability of the assay to
measure accurately PPV infectivity in the pre-
sence of another virus. Reovirus was chosen in
this study since it is a non-enveloped virus and is
propagated in a similar medium as PPV. The
PPV assay was conducted to measure PPV
concentrations in the presence and absence of
reovirus and the results were compared.
Matrix effect‘‘It may be important to consider the variability
of the matrix due to the physiological nature of
the sample’’ and ‘‘appropriate steps should be
taken to ensure the lack of matrix effects
throughout the application of the method’’ [2].
This assay is used in determining PPV titers in
samples of different matrices, depending on the
protein purification processes in production. It is
required that the matrix effect be determined
during specific virus clearance studies [8,9]. In
this study, the matrix effects of three general
matrices, HBSS (Hank’s Balanced Salt Solutions)
used to dilute PPV during titrations and
the media that are used to propagate PPV
(Dulbecco’s Modified Eagle Medium (DMEM)
plus 2% fetal bovine serum (FBS) and DMEM
plus 10% FBS) were determined.
Stability‘‘Drug stability in a biological fluid is a function
of the storage conditions, the chemical proper-
ties of the drug, the matrix, and the container
system’’ [2]. The stability of PPV in three
matrices tested in this study was not determined
in this study; rather it was included in a separate
stability study.
RobustnessRobustness is the measure of an assay’s capacity
to remain unaffected by small, but deliberate,
variations in the method parameters. The
robustness of a method provides a good indica-
tion of the reliability of the method under
normal assay conditions. The variations to be
applied to the PPV assay are reagents, FBS, cell
density, cell passage number, adsorption tem-
perature and time, and incubation temperature.
Viral titers obtained at the low and high limits of
the variations were analyzed to determine if
there were any significant differences between
the results.
Assay validation results
The actual validation results for repeatability,
intermediate precision, accuracy, and linearity
are shown in Table 1. This was a full assay
validation study and the assay validation results
demonstrated that this assay is precise, accurate,
and linear, and robust, meeting the intended use
in pathogen safety validation studies to support
IND, BLA and commercial production of
biological products [10].
General Considerations forImplementation of GLP Requirements
Although the GLP regulations were initially
promulgated primarily for toxicology studies,
they are broad and comprehensive enough to
provide a compliance framework to ensure the
integrity and validity of other types of studies
96 H Li et al.
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[10]. For this reason, many companies choose to
implement GLP elements in their assay valida-
tion studies. GLP concepts, such as the require-
ments for protocol, final report, and conduct of
a nonclinical laboratory study including doc-
umentation practices can be directly applied to a
GLP-compliant assay validation study. For ex-
ample, the following compliance practices
should be implemented:
* A scientist or professional is designated as a
study director by management who is
responsible for ‘‘the technical conduct of the
study, as well as for the interpretation,
analysis, documentation, and reporting
of the results’’ as defined in 21 CFR Part
58.33.* All study personnel, including the study
director, should have appropriate training
and/or experience, which are supported by
training records.* A specific, detailed description of the bioana-
lytical method to be validated should be
written. In the Standard Operating Procedure
(SOP) for the PPV assay, the key assay
parameters were described. The operating
ranges of these parameters were clearly
defined, providing the basis for the robustness
assessment.* Applicable elements for a study protocol as
described in 21 CFR Part 58.120 should be
incorporated into the assay validation protocol.* There also should be SOPs for operation of
instruments. For example, SOPs were pre-
pared for biological cabinets, cell incubators,
and pipettes.* Good documentation practices should be
followed and sufficient records generated to
allow reconstruction of the experiments.
During the PPV assay validation, standard
forms were used to record the experimental
procedures.* Equipment/Computerized Systems: GLP reg-
ulations for equipment should be followed,
including validation of computerized data
collection systems.* All experiments used to make claims or
draw conclusions about the validity of the
method should be presented in the final
report.* Applicable requirements for a study report as
described in 21 CFR Part 58.185 should be
met when preparing the assay validation
study report.* After the completion of the assay validation
study, all raw data, the protocol, the final
report, and other documentation should be
archived.
The QAU fulfils its responsibilities as defined
in the GLP regulations in 21 CFR Part 58.35. To
monitor a study, the following audits are usually
performed: (1) protocol review; (2) critical
phase/‘in-life’ audits (required); and (3) final
report audit (required). The frequency of critical
phase inspection should be determined based on
the nature of the assay validation study. Some
points for consideration when conducting
quality auditing are discussed in detail in the
following sections.
However, there are challenges in implement-
ing GLP compliance practices in assay valida-
tion studies. The following are some scenarios
that require careful interpretation of the GLP
regulations:
* The definitions for test article and test system
are not directly applicable because the pur-
pose of this type of study is to validate an
assay and not to test the safety of a drug or a
biological product. When entering the study
Table 1. Assay validation results
Assay parameter Result Acceptance criteria
Repeatability (% CV) 7.5% 25%Intermediate precision (% CV) 6.5% 25%Accuracy (recovery) 105% 100�30%Linearity (R2) 0.99 >0.95
GLP-compliant Assay Validation Studies 97
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information on the master schedule, it should
be indicated as such.* Reference standards are critical to a validation
study. The purity of the reference standard
used to prepare spiked samples can affect
study data. Also, if possible, the reference
standard should be identical to the analyte.
When this is not possible, an established
chemical form (free base or acid, salt or ester)
of known purity can be used [2]. There are
three types of reference standards: ‘‘(1)
Certified reference standards (e.g., United
States Pharmacopoeia compendial standards);
(2) Commercially supplied reference stan-
dards (from a reputable commercial source);
(3) Other materials of documented purity
custom-synthesized by an analytical labora-
tory or other noncommercial establishment.
The source, lot number, expiration date,
certificate of analyses when available, or
internally or externally generated evidence of
identity and purity should be furnished for
each reference standard’’ [2]. In the PPV assay
validation study, an in-house PPV stock was
characterized and used.* A bioanalytical method should be validated
for the intended use or application. A
protocol should include the acceptance criter-
ia for method parameters, which are estab-
lished based upon the application of the
assay. Remedial conditions for not meeting
such validation standards or revalidation
requirements should be provided. For exam-
ple, if the method does not meet the pre-
determined acceptance criteria, the method
parameters need to be readjusted and revali-
dated or the application is limited. The results
of the PPV assay validation study demon-
strated that the assay is suitable for use in
viral clearance safety studies.
Points for Consideration for AuditingMethod Validation Studies
Expectations for QAU
To be proactive and effective, QAU auditors
should be familiar with assay validation guide-
lines and requirements. The QAU may be asked
to provide guidance on designing assay valida-
tion studies. This is especially the case when the
QAU is helping a new group in either an
industrial or academic setting to establish a
GLP compliance program. The analytical scien-
tists may not necessarily understand the method
validation requirements. The QAU not only
performs the function of monitoring the com-
pliance of studies with GLPs, but also provides
assistance in the implementation of necessary
practices for compliance with the GLP regula-
tions as well as with regulatory guidelines on
method validation.
A good understanding of assay procedures
and validation requirements is very helpful for
auditors to conduct inspections. According to
the OECD GLP requirements, ‘‘[I]ndividuals
appointed to QA functions should have the
ability to understand the basic concepts under-
lying the activities being monitored’’ [11]. This
understanding also helps QAU auditors, as a
partner in drug development, to take a ‘risk-
based’ approach to auditing, knowing which
elements are most critical to data accuracy [12].
One way to follow new regulatory develop-
ments is to collect and analyze assay validation
related citations in regulatory inspections such
as FDA 483 s, Establishment Inspection Reports
and Warning Letters. It is very helpful to identify
common pitfalls encountered during assay vali-
dation studies, which are key reminders for QA
auditors to keep an eye on these critical issues.
The following are a few examples of FDA 483
citations on assay validation studies. They
pointed to the importance of reference standard,
meeting acceptance criteria, and matrix effect.
* ‘‘ The accuracy and sensitivity of the X and Y
assays were not determined in that the purity
of the reference material had not been
determined.’’ [13]* ‘‘Assay runs were accepted even though they
failed one or more predetermined acceptance/
rejection criteria.’’ [13]* ‘‘ The matrix that the assay was validated
at the contract research organization was
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different from the matrix of the samples from
the Sponsor.’’ [13]
Reviewing protocol
Reviewing a draft assay validation protocol
provides the first opportunity for a QA auditor
to understand and evaluate the method, the
scope of validation study and the strategies that
the study director plans to take to complete the
method validation. The following is a list of
reminders for auditors, including some key areas
to focus on during an audit of an assay
validation protocol. This list is not intended to
be exclusive, and not all elements apply to every
study:
* Be familiar with the bioanalytical method and
the instruments involved* Be familiar with assay validation guidelines* Verify that the method and applicable equip-
ment SOPs are written* Ensure that computerized systems are vali-
dated* Verify the assay validation design against
regulatory guidelines* Ensure that reference standards are appro-
priate and properly documented* Verify that statistical terminology and calcu-
lations to be used are described in the
protocol* Verify there are the acceptance criteria for
each method parameter to be validated* Ensure that the acceptance criteria are satis-
factory to meet the intended use of the
method* Verify that the matrix effect is evaluated* Verify that stability of samples are assessed
Auditing final report
The QA unit is required to audit the final assay
validation report to ensure validity and quality
of the data. The following is a list of key areas
that warrant a close look during a final report
audit:
* Evaluate the execution of the protocol
including documentation
* Verify that experiments are conducted
according to the protocol* Verify number of experiments* Evaluate use and handling of reference
standards* Assess handling of reagents and samples* Check statistical calculations* Verify if the acceptance criteria meet the
intended use of the assay* Verify if the acceptance criteria are met* Check handling of out-of-specification results* Verify if repeats are justified* Ensure that matrix effects are evaluated* Verify if revalidation requirements are in-
cluded
Conclusions
GLP-compliant assay validation studies present
a unique challenge to QAU auditors. To be
effective in performing QA functions when
monitoring GLP-compliant method validation
studies, QA auditors should have a good under-
standing of analytical procedures and regulatory
requirements for method validation. This will
help QA auditors efficiently navigate in the large
amount of complex data and not lose sight of
key compliance areas when conducting audits.
Acknowledgements
Authors thank Kang Cai and Randal
Hartwell for reviewing the manuscript.
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